Nanomedicine with eNOS-like enzyme activity, preparation method and application thereof

Abstract

The present application relates to a kind of nano-drugs with eNOS-like enzyme activity and its preparation method and application, belong to nano-drug technical field.Solve the technical problems that drug cannot pass through blood-brain barrier and cannot simultaneously inhibit neuron calcium overload, reduce mitochondrial damage and endoplasmic reticulum stress, inhibit inflammatory storm and other multiple effects in prior art.The nano-drug of the present application is the NO embedded molybdenum sulfide nanomaterial obtained by introducing S-nitroso group on the surface of small particle molybdenum sulfide.The nano-drug prepared by the present application has super strong antioxidant activity and stable NO release capacity, can effectively target CIRI brain tissue damage and stable release pure NO, can effectively treat cerebral ischemia-reperfusion injury, while improving intracellular Ca 2+ Overload, oxidative stress and inflammatory storm, can effectively protect neuron mitochondria from overload Ca 2+ And mtROS Damage, and significantly restore mitochondrial function in CIRI and other advantages.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanomedicine, in particular to a kind of nano-drug with eNOS enzyme activity and its preparation method and application. BACKGROUND

[0002] Cerebral infarction (CI) is the first cause of long-term disability worldwide, with more than 13.7 million new patients and more than 5.8 million deaths related to it each year. Early recanalization of occluded cerebral vessels using tissue plasminogen activator (tPA) or endovascular thrombectomy is the only intervention approved by the US Food and Drug Administration (FDA) for the treatment of CI. Unfortunately, only less than 5% of patients can benefit from these treatments, mainly due to the fact that recanalization beyond the therapeutic time window can further induce cerebral ischemia-reperfusion injury (CIRI), exacerbating the pathological process of CI. Therefore, there is an urgent need for new strategies to prevent and treat CIRI.

[0003] The pathological mechanism of CIRI is complex, mainly involving neuronal apoptosis caused by calcium overload, oxidative stress, and inflammatory storm. Specifically, the massive release of glutamate during CIRI induces neuronal intracellular Ca 2+ flow through postsynaptic N-methyl-D-aspartate receptors (NMDARs) leads to mitochondrial dysfunction, mitochondrial reactive oxygen species (mtROS) burst, and subsequent release of pro-apoptotic factor cytochrome C (Cyt-C) and endoplasmic reticulum stress (ERS), ultimately leading to neuronal damage and death. In addition, the release of mitochondrial deoxyribonucleic acid (mtDNA) from damaged neurons can activate the cyclic guanosine monophosphate-adenosine monophosphate synthase-interferon gene stimulator (cGAS-STING) signaling pathway in microglia, inducing microglia to polarize to a pro-inflammatory phenotype (M1), triggering a strong inflammatory storm and exacerbating neuronal death. Ultimately, calcium overload, oxidative stress, and inflammatory storm promote each other, forming a vicious cycle of neuronal damage and death. Therefore, the therapeutic effect of a single target is limited and short-lived, and the development of a drug platform that can simultaneously reverse calcium overload, oxidative stress, and inflammatory storm is crucial for the treatment of CIRI. However, due to the complex pathological mechanism of CIRI, there is currently no research that can simultaneously reverse these malignant progressions.

[0004] NO is a supermolecule with multiple beneficial biological effects, especially in cerebrovascular diseases. The loss of NO homeostasis plays a fundamental role in CIRI. NO mainly comes from three enzymes: eNOS from vascular endothelial cells, nNOS from neurons, and iNOS from glial cells or macrophages. Physiologically, at low reactive oxygen species (ROS) levels, NO mainly comes from eNOS and nNOS at appropriate concentrations, which can inhibit thrombosis, improve ischemic collateral circulation, and inhibit inflammatory response. More importantly, NO can inhibit Ca 2+ influx by inhibiting related protein kinases and calcium-derived nNOS-mediated neurotoxicity to alleviate intracellular Ca 2+ overload. However, under CIRI pathological conditions, eNOS and nNOS no longer generate NO, but superoxide (O2 .- ) and other ROS, because the NADPH-donated electrons are "uncoupled" from the activity of nitric oxide synthase (NOS) and the availability of L-arginine / BH4 is reduced. At the same time, M1-polarized microglia produce a large amount of NO through iNOS, which is easily reacted with high concentrations of ROS (such as O2 .- ) in CIRI to form highly toxic peroxynitrite (ONOO - ), further exacerbating neuronal damage and death. In recent years, emerging NO-based treatment methods are also developing, including NO donor S-nitrosoglutathione (GSNO), direct inhalation of NO gas, NO nanocarriers, etc. However, NO-based treatment methods have not achieved ideal therapeutic effects in the treatment of CIRI. On the one hand, the release rate of NO is difficult to control, and too high or too low NO concentration can have harmful effects on CIRI; on the other hand, NO cannot remove ROS in situ, but instead promotes the generation of destructive ONOO - , which in turn exacerbates neuronal damage and death. Therefore, solving the sustained and "pure" release of NO is a key problem that needs to be solved for NO-related therapy to treat CIRI. SUMMARY

[0005] The present application solves the technical problem in the prior art that drugs cannot cross the blood-brain barrier and cannot simultaneously inhibit neuronal calcium overload, reduce mitochondrial damage and endoplasmic reticulum stress, and inhibit inflammatory storm, and provides a nano-drug with eNOS-like enzyme activity and a preparation method and application thereof.

[0006] To solve the above technical problems, the technical scheme of the present application is as follows:

[0007] A nano drug with eNOS-like enzyme activity, which is a NO-embedded molybdenum sulfide nano material (MSNO) obtained by introducing S-nitroso (-SNO) groups on the surface of small particle molybdenum sulfide (MS); the S-nitroso groups are double-connected to the surface of molybdenum sulfide through S-NO bonds and Mo-N coordination bonds.

[0008] In the above technical solution, preferably, the particle size of the small particle molybdenum sulfide ranges from 20 to 200 nm.

[0009] In the above technical solution, preferably, the introduction of S-nitroso (-SNO) groups on the surface of small particle molybdenum sulfide (MS) is obtained by reacting small particle molybdenum sulfide (MS) with a chemical drug containing S-nitroso (-SNO) groups.

[0010] In the above technical solution, further preferably, the chemical drug containing S-nitroso (-SNO) groups is sodium nitrite or sodium nitroprusside, etc.

[0011] A preparation method of a nano drug with eNOS-like enzyme activity, comprising the following steps:

[0012] Ammonium molybdate ((NH4)6Mo7O 24 ·4H2O) and thiourea (CN2H4S) are prepared into MS by a hydrothermal method; small particle MS is screened out by differential centrifugation;

[0013] The small particle MS is washed with hydrochloric acid and reacts with a chemical drug containing S-nitroso (-SNO) groups to introduce S-nitroso (-SNO) groups on the surface of MS to obtain NO-embedded MSNO.

[0014] In the above technical solution, preferably, the reaction system for preparing MS is an alkaline buffer solution with pH=8-10, the reaction temperature is 180℃, and the reaction time is 7-24 hours; the reaction system for introducing S-nitroso (-SNO) groups is 0℃, and the reaction time is 12-24 hours.

[0015] In the above technical solution, preferably, the mass ratio of ammonium molybdate and thiourea is between 5:1 and 1:5.

[0016] In the above technical solution, preferably, the rotation speed of the differential centrifugation method for screening out small particle MS is between 1000 rpm and 5000 rpm, and the centrifugation time is between 1 min and 10 min; when washing the small particle MS, the concentration of hydrochloric acid ranges from 6 mol / L to 12 mol / L, and the washing time is 12-24 h.

[0017] In the above technical solution, preferably, the mass ratio of the MS and the chemical drug containing the S-nitroso (-SNO) group is between 10:1 and 1:10.

[0018] The application of the nano drug with eNOS-like enzyme activity in the preparation of a drug for treating cerebral ischemia-reperfusion injury.

[0019] The beneficial effects of the present application are:

[0020] The nano drug with eNOS-like enzyme activity of the present application is prepared by further introducing S-nitroso (-SNO) on the surface of small particles MS, and has super strong antioxidant activity and stable NO release capacity. Figure 1 )。

[0021] The nano drug with eNOS-like enzyme activity of the present application is prepared by a simple and green synthesis method, and the raw materials used are cheap and easy to obtain.

[0022] The nano drug with eNOS-like enzyme activity of the present application can effectively target the damaged brain tissue during CIRI and stably release pure NO. Figure 2 )。

[0023] The nano drug with eNOS-like enzyme activity of the present application can effectively treat cerebral ischemia-reperfusion injury. Figure 3 )。

[0024] The nano drug with eNOS-like enzyme activity of the present application can simultaneously improve the intracellular Ca 2+ overload, oxidative stress and inflammatory storm induced by CIRI. Figure 4 )。

[0025] The nano drug with eNOS-like enzyme activity of the present application can effectively protect the mitochondria of neurons from the damage of overloaded Ca 2+ and mtROS, and significantly restore the mitochondrial function in CIRI. Figure 5 )。

[0026] The nano drug with eNOS-like enzyme activity of the present application can effectively reduce the release of mitochondrial DNA (mtDNA) of neurons to the external space, inhibit the activation of the cGAS-STING signal of microglial cells, and thus reverse the neuroinflammation induced during CIRI. Figure 6 )。

[0027] The nano drug with eNOS-like enzyme activity of the present application can effectively alleviate the endoplasmic reticulum stress of neurons associated with mitochondrial damage, and significantly reduce the apoptosis of neurons. Figure 7 )。

[0028] The nano drug with eNOS-like enzyme activity has good biocompatibility, is non-toxic to hippocampal neuron cells (SH-5Y5Y cells) and microglial cells (BV2 cells) at a drug concentration involved in the present application, and has no effect on the heart, liver, spleen, lung, kidney and brain of normal rats after long-term administration. Figures 8-10 )。 BRIEF DESCRIPTION OF DRAWINGS

[0029] The present application will be further described in detail below in combination with the drawings and specific embodiments.

[0030] Figure 1 The results of synthesis and characterization of the nano drug 1 (MSNO) synthesized in Example 1 are shown in the figures. Among them, a is a schematic diagram of the synthesis of MSNO; b is a transmission electron microscope (TEM) visualization diagram of MSNO; c is a result diagram of the hydrodynamic diameter detection of MSNO; d is an FT-IR spectrum diagram of MSNO and MS; e is an XPS spectrum diagram of Mo 3p of MS and Mo 3p+N 1s of MSNO; f is an XPS spectrum diagram of S2p of MS and MSNO; g is a NO release behavior diagram of MSNO and GSNO under the same dose (1 mg / mL); h is a diagram of the O2 .- elimination ability of MSNO; i is a diagram of the ONOO .- generation ability of GSNO, GSNO+O2 .- , MS+O2 .- , MSNO+O2 - ; j is a diagram of the ·OH elimination ability of MSNO.

[0031] Figure 2 The results of the CIRI ischemic brain tissue targeting and brain tissue NO level detection of the nano drug 1 (MSNO) synthesized in Example 1 are shown in the figures. Among them, a is a schematic diagram of BBB damage during CIRI; b is a MCAO model diagram; c is a TEM diagram of brain tissue capillary; d is a statistical diagram of I / R brain tissue capillary endothelial gap; e is a representative image of bright field and fluorescence imaging of the brain, heart, lung, liver, spleen and kidney of rats in the Sham group and the I / R group after injection of FITC-MSNO for 1 h; f is a diagram of Mo content in the brain tissue of rats in the Sham group and the I / R group detected by ICP-MS at different time points after intravenous injection of MSNO; g-h are representative images (g) and quantitative statistical diagrams (h) of NO level in the cerebral infarction area detected by small animal imaging technology at different time points; i-j are representative images (j) and quantitative statistical diagrams (i) of ONOO - level in the brain tissue; k-l are diagrams of the changes of systolic pressure (k) and diastolic pressure (l) with time after administration of equal doses of GSNO and MSNO to SD rats.

[0032] Figure 3Figure for the improved efficacy results of the nanodrug 1 (MSNO) synthesized in Example 1 in CIRI. Among them, a is the flow chart of the efficacy study of MSNO in CIRI; b is the quantitative statistical diagram of TTC staining of CIRI rats treated with different doses of MSNO; c-d are representative images (c) and quantitative statistical diagram (d) of TTC staining of brain tissue of CIRI rats treated with NAC, GSNO, MS, MSNO at a dose of 0.5 mg / kg; e is the neurological score diagram of brain tissue of CIRI rats treated with NAC, GSNO, MS, MSNO at a dose of 0.5 mg / kg; f is a representative image of Nissl staining of brain tissue of rats in different treatment groups (red arrow marks atrophied neurons, black arrow marks tissue vacuolization); g is a representative image of HE staining of brain tissue in different treatment groups.

[0033] Figure 4 Figure for the mechanism exploration results of the nanodrug 1 (MSNO) synthesized in Example 1 to improve CIRI. Among them, a is the correlation heat map between samples in each group; b is the VENN / UpSetR graphical analysis showing the results of differentially expressed genes between Sham group, I / R group and MSNO treatment group; c is the volcano plot depicting the differentially expressed genes between MSNO group and I / R group brain tissue; d is the GO enrichment analysis diagram of biological processes involved in differentially expressed genes between I / R group and MSNO treatment group; e-j are visualization heat maps of differentially expressed genes in biological processes involved in GO enrichment analysis, including calcium signaling pathway (e), response to inflammation (f), mitochondrial function (g), ROS response and regulation (h), ERS (i), and apoptosis process (j); k is the KEGG pathway enrichment analysis diagram of DEG of MSNO treatment group and I / R group.

[0034] Figure 5 Figure for the effective protection of the nanodrug 1 (MSNO) synthesized in Example 1 on mitochondria from damage by Ca 2+ and mtROS, and significant recovery of mitochondrial function in CIRI. Among them, a-b are representative images (a) and quantitative statistical diagram (b) of Ca 2+ content in SH-SY5Y cells in different treatment groups; c is a diagram of flow cytometry analysis of the percentage of DCFH-DA (ROS fluorescent probe) positive cells in each treatment group; d is a representative image of FITC-MSNO and mitochondria and co-localization analysis; e is a representative image of mtROS in SH-SY5Y cells in each group; f-g are representative images (f) and quantitative analysis diagram (g) of JC-1 staining to evaluate the integrity of MMPs in each group of cells; h is an ATP production assay in SH-SY5Y cells; i is a representative fluorescence image of ROS level in brain tissue of rats in each group; j-l are TEM images of brain tissue neurons mitochondria in Sham group (j), I / R group (k), MSNO treatment group (l).

[0035] Figure 6 The results of the inhibition of microglial cGAS-STING signal activation by nanodrug 1 (MSNO) synthesized in Example 1 to reverse the neural inflammation induced by CIRI are shown in the figures. Among them, a is the dsDNA / Tom20 / DAPI immunofluorescence staining image of the cerebral infarction area of each treatment group, and the white arrow indicates mtDNA; b is the result of immunofluorescence staining of iNOS (M1) and CD206 (M2) to detect the phenotype of microglial cells in the infarction area; c is the expression of cGAS in the brain infarction tissue of each treatment group analyzed by immunohistochemistry; d is the expression of STING in the brain infarction tissue of each treatment group analyzed by immunohistochemistry; e-i are the expression levels of inflammation-related proteins in brain tissue homogenate detected by WB method (cGAS (f), STING (g), P-IRF3 / IRF3 (h), P-P65 / P65 (i)) and the representative graph (e) is drawn; j-o are brain tissue inflammation factor detection graphs, including pro-inflammatory factors IL-1β (j), IL-6 (k), iNOS (l) and TNF-α (m), and anti-inflammatory factors IL-4 (n) and IL-10 (o); p is a mechanism diagram of MSNO improving neural inflammation by inhibiting microglial cGAS-STING pathway.

[0036] Figure 7 The results of the inhibition of endoplasmic reticulum stress and apoptosis in neurons by nanodrug 1 (MSNO) synthesized in Example 1 are shown in the figures.

[0037] Among them, a-c are brain neuron ER transmission electron microscope photos of Sham group (a), I / R group (b), and MSNO treatment group (c) rats, and the yellow arrow indicates ER; d is a schematic diagram of ERS and mitochondrial damage cascade; e-h are qPCR detection of brain tissue ERS-related factor expression level graphs, including Bip (e), PERK (f), ATF-6 (g), and IRE-1α (h); i-j are brain tissue TUNEL staining (j) and quantitative analysis (i); k-o are WB detection of the expression levels of apoptosis-related proteins in brain tissue homogenate (Bax (k), bcl-2 (l), C-Cas3 / Cas3 (m), C-Cyt C (n)) and the representative graph (o) is drawn. p-q are flow cytometry analysis (q) and quantitative analysis results of SH-SY5Y cell apoptosis in different treatment groups (p).

[0038] Figure 8The results of the in vivo biological safety evaluation of the nanodrug 1 (MSNO) synthesized in Example 1 are shown in the figures. Among them, a is the HE staining figure of the main organs (heart, liver, spleen, lung, kidney and brain) of normal rats and rats 24 hours after injection of MSNO; b-e are the detection results of liver function (alanine aminotransferase (b) and gamma-glutamyl transferase (c)) and kidney function (creatinine clearance (d) and blood urea nitrogen (e)) of rats 24 hours after sublingual injection of MSNO; f-s are the blood routine examination results (24 hours after sublingual injection of MSNO), including white blood cell count (f), neutrophil count (g), lymphocyte count (h), monocyte count (i), neutrophil percentage (j), monocyte percentage (k), red blood cell count (l), hemoglobin content (m), hematocrit (n), mean corpuscular hemoglobin content (o), red blood cell distribution width variation coefficient (p), platelet count (q), mean platelet volume (r), and platelet distribution width (s).

[0039] Figures 9-10 The results of the in vitro biological safety evaluation of the nanodrug 1 (MSNO) synthesized in Example 1 are shown in the figures, wherein, Figure 9 The effect of MSNO of different concentrations on the cell viability of SH-SY5Y cells after 24h and 48h of treatment is shown in the figure. Figure 10 The effect of MSNO of different concentrations on the cell viability of BV2 cells after 24h and 48h of treatment is shown in the figure. DETAILED DESCRIPTION

[0040] The inventive idea of the present application is that NO has been identified as one of the most functional and unique molecules in the human body, and its protective effect in cerebrovascular diseases has also been widely verified. However, the beneficial effect of NO depends on its concentration and ROS level, which makes it difficult for current NO donors to treat CIRI. Based on this, the present application aims to develop a customized MoS2-based NO donor (MSNO) that connects the nitroso group to the two-dimensional plane of MoS2 through S-NO bond and Mo-N coordination bond to enable it to release NO stably like eNOS enzyme and avoid the generation of highly toxic ONOO - After intravenous injection, MSNO with appropriate nanosize can penetrate the blood-brain barrier and effectively treat CIRI through multiple actions: inhibition of calcium overload, reduction of mitochondrial damage and endoplasmic reticulum stress, and inhibition of inflammatory storm. The present application opens up a new way for the treatment of CIRI and provides a promising method for other ischemia-reperfusion injury diseases.

[0041] The technical solutions of the present application are described below through examples, but it should be understood that the following examples do not limit the scope of protection of the present application. The reagents used in the examples are commercially available.

[0042] Example 1

[0043] (1) Synthesis of molybdenum sulfide (MS)

[0044] (NH4)6Mo7O 24 ·4H2O (18.5375 g) and CN2H4S (7.9925 g) were dissolved in 500 mL of deionized water, and the pH of the reaction system was detected to be about 9, and then transferred to a high-pressure reaction kettle for reaction at 180°C for 24 h. The reaction product was washed with ultrapure water for 3 times to remove other impurities, and MS was obtained.

[0045] (2) Screening of small particle MS

[0046] The sample obtained in step (1) was centrifuged at 2000 rpm for 4 min, and the precipitate was discarded to obtain small particle MS. The particle size of the small particle molybdenum sulfide was in the range of 20-200 nm.

[0047] (3) Synthesis of molybdenum sulfide embedded with NO (MSNO)

[0048] The sample MS (0.5 g) obtained in step (2) and 0.1 mL of concentrated hydrochloric acid (12 mol / L) were mixed and stirred for 24 h. The system was transferred to ice, and 1 mL of NaNO2 (0.1 g / mL) was added after pre-cooling for 10 min. The reaction was carried out in an ice bath for 7 days in the dark. The reaction product was washed with ultrapure water for 3 times to obtain the final product, which was named as nanodrug 1 and represented as MSNO.

[0049] Figure 1 b is the TEM visualization of the eNOS enzyme-like nanodrug prepared based on MS. As can be seen from the TEM picture, the particle size of the nanodrug is in the range of 60-100 nm.

[0050] Example 2

[0051] (1) Synthesis of molybdenum sulfide (MS)

[0052] (NH4)6Mo7O 24 ·4H2O (22.5 g) and CN2H4S (4.5 g) were dissolved in 500 mL of deionized water, and the pH of the reaction system was detected to be about 10, and then transferred to a high-pressure reaction kettle for reaction at 180°C for 24 h. The reaction product was washed with ultrapure water for 3 times to remove other impurities, and MS was obtained.

[0053] (2) Screening of small particle MS, which is the same as in Example 1.

[0054] (3) Synthesis of molybdenum sulfide embedded with NO (MSNO), which is the same as in Example 1. The obtained nanodrug is named as nanodrug 2.

[0055] Example 3

[0056] (1) Synthesis of molybdenum sulfide (MS)

[0057] (NH4)6Mo7O 24 • 4H2O (20.25 g) and CN2H4S (6.75 g) were dissolved in 500 mL of deionized water, the pH of the reaction system was detected to be about 9.5, and then transferred to a high-pressure reactor for reaction at 180°C for 24 h. The reaction product was washed with ultrapure water for 3 times to remove other impurities, and MS was obtained.

[0058] (2) Screening of small particle MS Same as Example 1.

[0059] (3) Synthesis of molybdenum sulfide embedded with NO (MSNO) Same as Example 1, and the obtained nanodrug is called nanodrug 3.

[0060] Example 4

[0061] (1) Synthesis of molybdenum sulfide (MS)

[0062] (NH4)6Mo7O 24 • 4H2O (13.5 g) and CN2H4S (13.5 g) were dissolved in 500 mL of deionized water, the pH of the reaction system was detected to be about 8.5, and then transferred to a high-pressure reactor for reaction at 180°C for 12 h. The reaction product was washed with ultrapure water for 3 times to remove other impurities, and MS was obtained.

[0063] (2) Screening of small particle MS Same as Example 1.

[0064] (3) Synthesis of molybdenum sulfide embedded with NO (MSNO) Same as Example 1, and the obtained nanodrug is called nanodrug 4.

[0065] Example 5

[0066] (1) Synthesis of molybdenum sulfide (MS)

[0067] (NH4)6Mo7O 24 • 4H2O (6.75 g) and CN2H4S (20.25 g) were dissolved in 500 mL of deionized water, the pH of the reaction system was detected to be about 8.3, and then transferred to a high-pressure reactor for reaction at 180°C for 24 h. The reaction product was washed with ultrapure water for 3 times to remove other impurities, and MS was obtained.

[0068] (2) Screening of small particle MS Same as Example 1.

[0069] (3) Synthesis of molybdenum sulfide embedded with NO (MSNO) Same as Example 1, and the obtained nanodrug is called nanodrug 5.

[0070] Example 6

[0071] (1) Synthesis of molybdenum sulfide (MS)

[0072] (NH4)6Mo7O 24 • 4H2O (4.5 g) and CN2H4S (22.5 g) were dissolved in 500 mL of deionized water, the pH of the reaction system was detected to be about 8, and then transferred to a high-pressure reaction kettle for reaction at 180 °C for 24 h. The reaction product was washed with ultrapure water for 3 times to remove other impurities, and MS was obtained.

[0073] (2) Screening of small particle MS

[0074] (3) Synthesis of molybdenum sulfide embedded with NO (MSNO) Same as Example 1, the obtained nanodrug is called nanodrug 6.

[0075] Example 7

[0076] (1) Synthesis of molybdenum sulfide (MS) Same as Example 1.

[0077] (2) Screening of small particle MS

[0078] Take the sample obtained in step (1), centrifugal screening at 1000 rpm for 4 min, and discard the precipitate to obtain small particle MS.

[0079] (3) Synthesis of molybdenum sulfide embedded with NO (MSNO) Same as Example 1, the obtained nanodrug is called nanodrug 7.

[0080] Example 8

[0081] (1) Synthesis of molybdenum sulfide (MS) Same as Example 1.

[0082] (2) Screening of small particle MS

[0083] Take the sample obtained in step (1), centrifugal screening at 3000 rpm for 4 min, and discard the precipitate to obtain small particle MS.

[0084] (3) Synthesis of molybdenum sulfide embedded with NO (MSNO) Same as Example 1, the obtained nanodrug is called nanodrug 8.

[0085] Example 9

[0086] (1) Synthesis of molybdenum sulfide (MS) Same as Example 1.

[0087] (2) Screening of small particle MS

[0088] Take the sample obtained in step (1), centrifugal screening at 4000 rpm for 10 min, and discard the precipitate to obtain small particle MS.

[0089] (3) Synthesis of NO-embedded molybdenum sulfide (MSNO) The synthesis of MSNO was the same as Example 1. The obtained nanodrug was named as nanodrug 9.

[0090] Example 10

[0091] (1) Synthesis of molybdenum sulfide (MS) The synthesis of MS was the same as Example 1.

[0092] (2) Screening of small particle MS

[0093] The sample obtained in step (1) was centrifuged at 5000 rpm for 4 min for screening, and the precipitate was discarded to obtain small particle MS.

[0094] (3) Synthesis of NO-embedded molybdenum sulfide (MSNO) The synthesis of MSNO was the same as Example 1. The obtained nanodrug was named as nanodrug 10.

[0095] Example 11

[0096] (1) Synthesis of molybdenum sulfide (MS) The synthesis of MS was the same as Example 1.

[0097] (2) Screening of small particle MS The synthesis of MS was the same as Example 1.

[0098] (3) Synthesis of NO-embedded molybdenum sulfide (MSNO)

[0099] The sample MS (0.54 g) obtained in step (2) was mixed with 0.1 mL of concentrated hydrochloric acid (12 mol / L) and stirred for 24 h. The system was transferred to ice, 1 mL of NaNO2 (0.06 g / mL) was added after pre-cooling for 10 min, and the reaction was carried out in an ice bath for 7 h in the dark. The reaction product was washed with ultrapure water for 3 times to obtain the final product, which was named as nanodrug 11.

[0100] Example 12

[0101] (1) Synthesis of molybdenum sulfide (MS) The synthesis of MS was the same as Example 1.

[0102] (2) Screening of small particle MS The synthesis of MS was the same as Example 1.

[0103] (3) Synthesis of NO-embedded molybdenum sulfide (MSNO)

[0104] The sample MS (0.3 g) obtained in step (2) was mixed with 0.1 mL of concentrated hydrochloric acid (12 mol / L) and stirred for 24 h. The system was transferred to ice, 1 mL of NaNO2 (0.3 g / mL) was added after pre-cooling for 10 min, and the reaction was carried out in an ice bath for 7 h in the dark. The reaction product was washed with ultrapure water for 3 times to obtain the final product, which was named as nanodrug 12.

[0105] Example 13

[0106] (1) Synthesis of molybdenum sulfide (MS) The synthesis of MS was the same as Example 1.

[0107] (2) Screening of small particle MS Same as Example 1.

[0108] (3) Synthesis of NO-embedded molybdenum sulfide (MSNO)

[0109] The sample MS obtained in step (2) (0.1 g) was mixed with 0.1 mL of concentrated hydrochloric acid (12 mol / L) and stirred for 24 h. The system was transferred to ice, 1 mL of NaNO2 (0.5 g / mL) was added after pre-cooling for 10 min, and the reaction was carried out in an ice bath for 24 h in the dark. The reaction product was washed repeatedly with ultrapure water for 3 times to obtain the final product, which was named as nano-drug 13.

[0110] Example 14

[0111] (1) Synthesis of molybdenum sulfide (MS) Same as Example 1.

[0112] (2) Screening of small particle MS Same as Example 1.

[0113] (3) Synthesis of NO-embedded molybdenum sulfide (MSNO)

[0114] The sample MS obtained in step (2) (0.06 g) was mixed with 0.1 mL of concentrated hydrochloric acid (12 mol / L) and stirred for 24 h. The system was transferred to ice, 1 mL of NaNO2 (0.54 g / mL) was added after pre-cooling for 10 min, and the reaction was carried out in an ice bath for 12 h in the dark. The reaction product was washed repeatedly with ultrapure water for 3 times to obtain the final product, which was named as nano-drug 14.

[0115] Example 15

[0116] The nano-drug 1 (MSNO) synthesized in Example 1 was taken as an example for structural characterization and evaluation of NO release and antioxidant performance of the nano-drug. The specific steps are as follows:

[0117] The NO detection kit (Bi Yun Tian) was used to determine the in vitro NO release. The NO released by MSNO and S-nitrosoglutathione (GSNO) is rapidly oxidized to NO2 - , which reacts with sulfanilamide to form a product with a maximum absorption peak at 540 nm. The MSNO solution (or GSNO solution) with a concentration of 1.0 mg / mL was placed in a 37°C water bath, and 1 mL of sample was taken out at 0 min, 1 h, 3 h, 6 h, 12 h, 24 h, 2 d, 3 d, 4 d, and 5 d. The supernatant was taken through a water filter (0.22 μm). Griess I and Griess II were added, respectively, and the absorbance of the mixture was measured at a wavelength of 540 nm by an enzyme marker.

[0118] Superoxide anion (O2.- )Clearance ability detection: The clearance ability of nano-drug 1 on O2 .- was detected by nitro blue tetrazolium (NBT) method. In the presence of methionine, riboflavin can be photoreduced to further generate O2 .- , O2 .- can reduce NBT to methylene blue, which has a maximum absorption at 560 nm. Briefly: 390 μL of methionine (0.1 M), 6 μL of riboflavin (20 μM), 23 μL of NBT (0.01 M), 1.5 mL of PBS (pH 7.4, 0.1 M), 1.1 mL of deionized water and 15 μL of MSNO solution at different concentrations (0 μg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.8 mg / mL, 1.6 mg / mL, 3.2 mg / mL) were mixed well and added to a quartz cuvette, which was placed on a UV / visible spectrophotometer to measure the baseline, then removed and irradiated under a UV lamp for 5 minutes. After irradiation, the UV absorption peak of the solution at 560 nm was detected, and the pseudo-SOD activity of the test sample was calculated. When the inhibition rate is 50%, the SOD enzyme activity in the test sample is recorded as 1 unit.

[0119] Hydroxyl radical (·OH) clearance ability detection: The clearance ability of MSNO on ·OH was detected by terephthalic acid fluorescence spectrophotometry. Ferrous sulfate and hydrogen peroxide catalyze the generation of ·OH through Fenton reaction, and non-fluorescent terephthalic acid is converted into fluorescent 2-hydroxy terephthalic acid. Briefly: PBS solution containing FeSO4 (0.5 mM), terephthalic acid (0.5 mM), H2O2 (1 mM) and different concentrations of MSNO (0 μg / mL, 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 4 μg / mL) was reacted at room temperature for 6 min, and then mixed well and added to a quartz cuvette. The fluorescence spectrophotometer detects the characteristic absorption peak of fluorescent 2-hydroxy terephthalic acid at 430 nm.

[0120] Peroxynitrite anion (ONOO - ) clearance ability detection: The clearance ability of MSNO on ONOO - was detected by pyrogallol red method. Pyrogallol red has a specific absorption peak at 540 nm, and ONOO - can quench pyrogallol red and reduce its specific absorption peak. Method: 0.1 M methionine (390 μL), 20 μM riboflavin (6 μL), 0.1 M (pH 7.4) PBS (1.5 mL), 1.1 mL of ultrapure water were added to the cuvette in turn and mixed well, then exposed to UV light for 5 min to generate O2 ·-The solution was transferred into a centrifuge tube, 24 μL (1 mg / mL) of ultrapure water / GSNO / MS / MSNO and an equal dose of pure GSNO solution were added respectively, and after incubation at 37 °C for 12 h, 10 μL of pyrogallol red solution was added, and the characteristic absorption peak of pyrogallol red at 540 nm was measured after reaction at room temperature for 20 min. The control sample was a pyrogallol red solution without ultraviolet irradiation and without drugs.

[0121] The results are shown in Figure 1 . Among them, it can be known from a in Figure 1 that MSNO is prepared by introducing a nitroso group on the surface of MS; it can be known from b in Figure 1 that MSNO is a three-dimensional flower-like structure composed of many nanosheets, and the MSNO nanosheet is observed to be derived from the (002) crystal plane of MoS2 by high-resolution TEM, and the interlayer spacing is It can be known from c in Figure 1 that the hydrodynamic average particle size of MSNO is 84.49 nm respectively; it can be known from d in Figure 1 that MSNO and MS both contain Mo-S stretching vibration at 591 cm -1 and 932 cm -1 . Different from MS, MSNO has characteristic peaks at 695 cm -1 and 1576 cm -1 , which are respectively attributed to S-N and N=O stretching vibration; it can be known from e in Figure 1 that N1s in MSNO is mainly attributed to N-O bond at 401.7 eV and Mo-N bond at 398.8 eV, indicating that the nitroso group in MSNO can form a coordination bond with adjacent Mo, thereby achieving slow release of NO; it can be known from f in Figure 1 that the S-H bond at 164.2 eV in MSNO is significantly reduced compared with MS, indicating that -SH is successfully converted to -SNO in the preparation process of MSNO; it can be known from g in Figure 1 that NO in GSNO is completely released in only 10 min, indicating that GSNO exhibits burst NO release behavior. In sharp contrast to GSNO, MSNO slowly releases NO within 120 hours, indicating that the coordination bond between NO and Mo in MSNO greatly slows down the self-splitting rate of -SNO in aqueous solution. In addition, the NO release curve of MSNO shows an approximate linear correlation between NO concentration and time within the first 24 hours, indicating that MSNO has controllable NO release behavior, and the stable state of NO concentration can be easily maintained by optimizing the dose of MSNO; it can be known from h in Figure 1 that MSNO inherits the strong O2 .- scavenging ability of MS, and its pseudo-SOD activity is as high as 105 U / mg; it can be known from i in Figure 1 that the O2.- The NO produced by GSNO generates a large amount of ONOO - in the system, while the ONOO - generated by the MSNO group is significantly less than the GSNO group, only 1 / 22 of the GSNO group; from Figure 1 It can be known from j that MSNO inherits the scavenging ability of MS to ·OH. In summary, from Figure 2 It can be known from the results that MSNO is successfully prepared, and MSNO has controllable NO release and antioxidant capacity.

[0122] Example 16

[0123] Taking the nanodrug 1 (MSNO) synthesized in Example 1 as an example, the targeting of the nanodrug to the ischemic brain tissue of CIRI rats and the NO level in the brain tissue were evaluated. The specific steps are as follows:

[0124] Fluorescence tracing of the drug distribution: the sham group (Sham group) and the CIRI group (I / R group) of rats were given the fluorescently labeled nanodrug 1 (MSNO-FITC) through the sublingual vein, and after 1 h, the brain, heart, liver, spleen, lung and kidney of the rats were taken and placed under a stereoscopic fluorescence microscope (Leica, M205FCA) to observe the distribution of the drug in the main organs of the rats and collect images.

[0125] ICP-MS detection of drug content in tissues: the Sham group of rats and the MSNO group of rats were injected with MSNO, and the brain, heart, liver, spleen, lung and kidney were collected at 1 h, 3 h, 6 h, 12 h and 24 h, respectively. The Mo element content of each organ was determined by ICP-MS method.

[0126] TEM of brain tissue: the brain tissue (less than 1 mm 3 ) of the Sham group, the I / R group and the MSNO group of rats was fixed with fresh TEM fixing solution overnight, and then the tissue was washed with PBS three times (15 minutes each time), and then replaced with 1% osmium acid mixture solution for 2 hours. After gradient dehydration, the tissue was embedded and solidified. The sample was cut into 60-80 nm thick sections using a microtome, stained with 3% uranyl acetate-citric acid lead, and observed for the morphology of neurons and the distribution of nanodrug 1 (MSNO) in mitochondria using a Jeol 1200EX transmission electron microscope.

[0127] NO in brain tissue: fresh brain tissue was taken from the Sham, I / R, GSNO, MS and MSNO groups of rats at 10 min, 30 min, 1 h and 6 h after drug injection, washed with PBS three times to remove surface blood, and then cut into 5 pieces vertically on the coronal plane. The brain tissue sections were soaked in DAF-FM DA staining solution, incubated at 37°C in the dark for 15 min, washed with PBS to remove residual dye, and then the images were collected using a small animal live optical imaging system.

[0128] The results are as follows Figure 2 As shown. Among them, by Figure 2 As can be seen from a, during CIRI, the BBB is affected by ROS bursts, pro-inflammatory cytokines, matrix metalloproteinases (MMPs), etc., and the tight junctions of capillary endothelial cells and the integrity of the basement membrane are disrupted, providing an opportunity for the specific enrichment of MSNO in CIRI lesions; Figure 2 As shown in b, the rat CIRI model simulates cerebral ischemia through middle cerebral artery occlusion (MCAO), and is established by removing the sutures blocking the middle cerebral artery after 2 hours of ischemia to restore blood supply for 24 hours; Figure 2 As shown in the middle c, compared with the Sham group, after 2 hours of ischemia, the vascular endothelial cells in the brain tissue of the I / R group were significantly swollen, and the tight junctions and basement membrane integrity were lost; Figure 2 As shown in the data, the average gap between tight junctions of capillary endothelial cells in the I / R group brain tissue was approximately 120 nm, providing an opportunity for smaller MSNO particles (approximately 85 nm) to penetrate the BBB gap and specifically target CIRI lesions. Figure 2 As shown in the data, FITC-MSNO in the Sham group was mainly distributed in the liver and kidneys, and almost none in the brain tissue of the Sham group and the healthy brain tissue of the I / R group. This is because the BBB in the healthy brain tissue prevents MSNO from entering. Conversely, in the I / R group rats, FITC-MSNO specifically targeted CIRI lesions after intravenous injection for only 1 hour, and the fluorescence intensity of FITC-MSNO in the CIRI lesions of the I / R group was 27 times that of the Sham group. Figure 2 As shown in the data, MSNO achieved targeted distribution in ischemic brain tissue 1 hour after intravenous injection, reaching a peak at approximately 6 hours (25 times that of the Sham group), followed by a gradual decrease in concentration. This indicates that MSNO can target ischemic brain tissue and be degraded. This evidence fully confirms that MSNO can specifically target CIRI tissue and is biodegradable; Figure 2As shown in the data from the gh study, in the MSNO group, NO was only distributed in the I / R brain tissue (right side of the yellow line) and maintained a relatively stable NO concentration after 1 hour, but was not distributed in the non-I / R brain tissue (left side of the yellow line). Unlike the MSNO group, in the GSNO group, the NO concentration in the non-I / R brain tissue was higher than that in the I / R brain tissue at 15 minutes. This interesting and contrasting phenomenon stems from the different physicochemical properties of MSNO and GSNO and the pathological characteristics of the BBB during CIRI. MSNO, as a large-size nanodrug, tends to accumulate in CIRI lesions through damaged BBB gaps. The distribution of GSNO is closely related to blood perfusion volume because GSNO is a small molecule. Since the blood supply to healthy brain tissue is better than that to CIRI lesions, the NO level in CIRI lesions in the GSNO group is lower than that in healthy brain tissue. Furthermore, due to the shorter half-life of GSNO in vivo, NO disappears more quickly in the GSNO group; Figure 2 From the information provided, we can see that the ONOO of the I / R group and the GSNO group... - Both were significantly elevated, being 6.21 times and 6.67 times higher than the Sham group, respectively. MS and MSNO significantly reduced ONOO. - This is because MS has a powerful ROS removal capability; by Figure 3 As far as we know, GSNO caused a sharp drop in blood pressure within 15 minutes in the GSNO group, with both systolic and diastolic blood pressure dropping to half of normal. Conversely, MSNO, due to its strong targeting of CIRI lesions and controllable NO release, had no significant effect on systemic blood pressure. In summary, from Figure 3 It is known that MSNO can target CIRI lesions with high specificity and can treat CIRI by clearing high concentrations of ROS and stably releasing "pure NO".

[0129] Example 17

[0130] Using the nanomedicine 1 (MSNO) synthesized in Example 1 as an example, the therapeutic effect of the nanomedicine on CIRI in rats was evaluated.

[0131] The specific steps are as follows:

[0132] TTC and neurological score: Male SD rats (8 weeks old, 260-280 g) were subjected to middle cerebral artery occlusion (MCAO) to mimic cerebral ischemia, and the occlusion wire was removed after 2 h of ischemia to restore blood supply for 24 h to mimic reperfusion. To evaluate the effect of drug intervention, MSNO was administered before reperfusion. After 24 h of reperfusion, the neurological function of the rats was evaluated by the neurological score standard. Subsequently, the rat brain tissue was taken and the infarct area of the rat brain was evaluated by triphenyltetrazolium chloride (TTC) staining. To evaluate the efficacy of the drug, the clinically approved antioxidant drugs NAC and GSNO and MS were used as controls. (The neurological score used the Zea Longa 5-point standard: 0 points represent no neurological deficit symptoms; 1 point represents an inability to fully extend the contralateral forelimb; 2 points represent turning to the paretic side when walking; 3 points represent the body tilting to the paretic side when walking; 4 points represent an inability to walk independently, and consciousness disorder; 5 points represent death.)

[0133] HE staining and Nissl staining: After 24 h of reperfusion, the rat brain tissue was fixed with 4% PFA for 24 h, dehydrated, and then embedded in paraffin blocks. The blocks were cut into 4 μm sections, and HE staining and Nissl staining were used to observe the histopathological changes in the tissues.

[0134] The results are shown in Table 1. Figure 3 Table 1 Figure 3 a is the treatment regimen of MSNO for treating CIRI; from Figure 3 b, it can be seen that even at an ultra-low therapeutic dose, MSNO can significantly reduce the cerebral infarction area, and the therapeutic effect increases in a dose-dependent manner. At the optimal dose (0.5 mg / kg), the cerebral infarction area treated with MSNO is reduced to 7.89%, which is much lower than that of the I / R group (44.58%); from Figure 3 c-d, it can be seen that the infarction area of the MSNO group is the smallest, which is much smaller than that of the MS group, the NAC group, the GSNO group, and the I / R group. MS has a certain therapeutic effect on CIRI due to its strong ROS scavenging ability, while the effects of NAC and GSNO are minimal; from Figure 3 e, it can be seen that the therapeutic effect of MSNO on improving the neurological function of I / R rats is much better than that of MS, NAC, and GSNO; from Figure 3 f, it can be seen that the number of Nissl staining spots (Nissl bodies) in the CIRI lesion of the I / R group is significantly reduced, and the morphology is reduced from regular tiger stripes to irregular dots or vacuoles. Both MS and MSNO treatment can improve the number and morphology of Nissl bodies, and the therapeutic effect of MSNO is significantly better than that of MS; from Figure 4 g, it can be seen that MSNO treatment can significantly improve the structure of the brain tissue. In summary, from Figure 4It can be seen that MSNO has a strong therapeutic effect on CIRI, and the effect is much better than MS, GSNO and NAC, which indicates that the efficacy of MSNO comes from its high targeting, ROS scavenging ability and controllable and precise release of NO on CIRI lesions.

[0135] Example 18

[0136] Taking the nano-drug 1 (MSNO) synthesized in Example 1 as an example, the possible mechanism of MSNO in treating CIRI was evaluated.

[0137] RNA sequencing analysis: Total RNA was extracted from the brain tissues of Sham, I / R and MSNO groups using TRIzol according to the manufacturer's protocol. Subsequent analysis and data mining were performed on Dr. Tom Multi-omics data mining system (https: / / biosys.bgi.com), using HISAT2 to map clean reads to the reference genome, and using Bowtie2 to align clean reads to gene sets.

[0138] The results are shown in Figure 4 . Among them, it can be known from a in Figure 4 that the Pearson correlation coefficients between 3 samples in each group are all greater than 0.94, indicating that the sample data consistency is high; it can be known from b in Figure 4 that VENN / UpSetR analysis found that there were 1937 differentially expressed genes (DEGs) between Sham and I / R groups, while there were only 99 DEGs between MSNO and Sham groups; it can be known from c in Figure 4 that compared with I / R group, 1647 genes were up-regulated and 790 genes were down-regulated in MSNO group; it can be known from d in Figure 4 that comparison of DEGs between Sham and I / R groups with gene ontology (GO) database found that the biological processes involved mainly included calcium ion channel, inflammatory response, mitochondrial function, oxidative stress, ERS and apoptosis; it can be known from e-j in Figure 5 that the gene expression of the above 6 biological processes in I / R group changed significantly compared with Sham group, while the gene expression in MSNO treatment group was close to that in Sham group, which fully confirmed the effective regulation of MSNO on the above 6 biological processes related genes; it can be known from k in Figure 5 that compared with I / R group, the DEGs of MSNO group were enriched in Ca 2+The relevant signal pathways, mitochondrial damage signal pathways (including ER function, cytokine-cytokine receptor interaction and apoptosis) and inflammation-related signal pathways (cytoplasmic DNA sensing pathway, tumor necrosis factor (TNF) signal pathway and NF-κB signal pathway) are strongly activated during CIRI, and MSNO can effectively inhibit the activation of these pathways. Therefore, MSNO can simultaneously improve the CIRI-induced intracellular Ca Figure 5 overload, oxidative stress and inflammation storm. 2+ overload, oxidative stress and inflammation storm.

[0139] Example 19

[0140] Example 1, to evaluate the results of the nanodrug effectively protecting mitochondria from damage by overload Ca 2+ and mtROS and significantly restoring mitochondrial function in CIRI. The specific steps are as follows:

[0141] Animal level: After 24h of reperfusion, the rat brain tissue was taken for frozen section, and the overall level of ROS in the brain tissue was evaluated by ROS probe (DHE) immunofluorescence staining.

[0142] Cell level: SH-SY5Y cells were seeded in 24-well plates at 1×10 4 / well, and after 24h, the SH-SY5Y cells were treated with medium containing (2 μg / mL) MSNO and 400 μM cobalt chloride for 16h, and then replaced with medium containing only 2 μg / mL MSNO for continued culture for 8h. The cells were incubated with ROS fluorescent probe (DCFH-DA), mitochondrial ROS fluorescent probe (MitoSox), and mitochondrial membrane potential detection fluorescent probe (JC-1) for 25min, washed twice with buffer, observed under a fluorescence microscope and images were collected; SH-SY5Y cells were seeded in 24-well plates at 1×10 4 / well, and after 24h, the SH-SY5Y cells were treated with medium containing (2 μg / mL) MSNO and 400 μM cobalt chloride for 16h, and then replaced with medium containing only 2 μg / mL MSNO for continued culture for 8h. An ATP detection kit was used to evaluate the ATP production content of SH-SY5Y cells.

[0143] The results are shown in Figure 5 . Among them, from a-b in Figure 5 , it can be seen that the intracellular Ca 2+ level of the H / R group of cells is 3.45 times that of the Normoxia group of SH-SY5Y cells, indicating that H / R causes severe calcium overload in SH-SY5Y cells. After MSNO treatment, the intracellular Ca 2+The level was close to that of the Normoxia group, and the effect was obviously better than that of MS. In addition to Ca 2+ In addition to overload, ROS burst is a key factor leading to mitochondrial damage in CIRI neurons; from Figure 5 As can be seen from Fig. 4c, by flow cytometry, the percentage of DCFH-DA positive cells in SH-SY5Y cells increased from 4.08% to 64.07% after H / R, while the percentage of DCFH-DA positive cells in cells treated with MS and MSNO decreased to 7.53% and 6.39%, respectively, indicating that MSNO and MS can effectively eliminate the ROS burst caused by H / R. The main source of ROS in neuronal cells is mitochondria, and targeting mitochondria by MSNO is the key to ROS elimination; from Figure 5 As can be seen from Fig. 4d, by co-labeling with Mito-tracker and FITC-MSNO, it was confirmed that MSNO efficiently targeted the mitochondria of SH-SY5Y cells under H / R conditions, and the Pearson coefficient was as high as 0.84; from Figure 5 As can be seen from Fig. 4e, H / R increased mtROS in SH-SY5Y cells by 3.36 times. As expected, MS and MSNO significantly reduced the level of mtROS in SH-SY5Y cells induced by H / R, and the level of mtROS was close to that of the normal group; from Figure 5 As can be seen from Fig. 4f-g, after H / R treatment, the proportion of normal MMP in SH-SY5Y cells decreased from 100% to 4.39%, while the proportion of normal MMP in the MSNO treatment group recovered significantly (80.26%); from Figure 6 As can be seen from Fig. 4h, MSNO effectively reversed the decrease in ATP production caused by H / R, and the effect was better than that of MS, indicating that MSNO can effectively restore the function of mitochondria damaged by H / R in SH-SY5Y cells; from Figure 6 As can be seen from Fig. 5i, the level of ROS in the brain tissue of the I / R group increased to about 3.4 times that of the Sham group, while the level of ROS in the CIRI lesion of the MS group and the MSNO group decreased to close to the level of the Sham group; from Figure 6 As can be seen from Fig. 5j-l, the mitochondria of neurons in the brain tissue of the I / R group were severely damaged, appearing swollen, dissolved, unclear ridges, and even vacuolization, while the mitochondrial structure recovered significantly after MSNO treatment, providing direct evidence for the protective effect of MSNO on mitochondria. In summary, from Figure 6 It can be seen that MSNO can effectively protect mitochondria from damage caused by Ca 2+ and mtROS overload, and significantly restore mitochondrial function in CIRI.

[0144] Example 20

[0145] As an example, the nano-drug 1 (MSNO) synthesized in Example 1 was used to evaluate the effect of the nano-drug on inhibiting the activation of cGAS-STING signal of microglial cells to reverse CIRI neuroinflammation. The specific steps are as follows:

[0146] Animal level: After 24 hours of reperfusion, the CIRI rats were taken for paraffin section of the rat brain tissue. The section was incubated with the primary antibody of dsDNA / Tom 20, iNOS, CD206 (Anti-dsDNA antibody, Abeam, AB27156; Tom20 Polyclonal antibody, Proteintech, 11802-1-AP; iNOS antibody, Affinity, AF0199; CD206 Recombant antibody, Proteintech, 81525-1-RR) at 4°C overnight, and the corresponding secondary antibody (Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, Invitrogen, A11029; Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488, Invitrogen, A11034) was added for 2 hours at room temperature. The section was observed under a fluorescence microscope. TM 488, Invitrogen, A11029; Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM555, Invitrogen, A21428) for 2 hours at room temperature. The slides were mounted with anti-fluorescence quenching solution containing DAPI, followed by acquiring amplified fluorescence images using LSM 900 equipped with Airyscan2 (Carl Zeiss). For quantification, for each brain tissue section, at least three random imaging fields within the lesion area were selected and analyzed using Image J; the expression levels of rat brain tissue inflammation-related proteins were detected. Specifically: CIRI rats were collected rat brain tissue by tissue homogenizer and separated proteins using RIPA (protease and phosphatase inhibitors) after 24h reperfusion. The protein concentration was analyzed by BCA kit, 30 μg total protein was separated by 8%-12% SDS-PAGE gel and transferred to PVDF membrane. The membrane was blocked with 5% milk in TBST for 1 hour at room temperature, and specific primary antibodies (cGAS, STING, P65, IRF3, P-IRF3, P-P65) were incubated overnight at 4°C. After that, the membrane was washed with TBST three times, then incubated with secondary antibody (Goat antimice IgG H&L (HRP); Goat anti Rabbit IgG H&L (HRP)) for 1 hour at room temperature. The bands were visualized by gel imaging system (Bio-Rad, USA) and quantified using ImageJ (V 1.53a) software; the expression levels of rat brain tissue inflammatory factors were detected. Specifically: CIRI rats were collected rat brain tissue by tissue homogenizer and the supernatant was detected by ELISA detection kit to detect the expression levels of brain tissue inflammatory factors, including pro-inflammatory factors interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and anti-inflammatory factors interleukin-4 (IL-4) and interleukin-10 (IL-10) after 24h reperfusion.

[0147] Cell level: Cell slides were placed in 24-well plates, then SH-SY5Y cells were seeded at 1 x 10 4 / Well seeded in 24-well plates, 24 hours later, SH-SY5Y cells were treated with medium containing 2 pg / mL MSNO-FITC + 400 pM cobalt chloride for 16 hours, then replaced with medium containing only 2 pg / mL MSNO-FITC for 8 hours. Cell slides were taken out, and incubated with dsDNA / Tom20 primary antibody (Anti-dsDNA antibody, Abeam, AB27156; Tom20 Polyclonal antibody, Proteintech, 11802-1-AP;) at 4°C overnight, and incubated with the corresponding secondary antibody (Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 488, Invitrogen, A11029; Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor TM 555, Invitrogen, A21428) at room temperature for 2 hours. The slides were mounted with anti-fluorescence quenching solution containing DAPI, and then the enlarged fluorescence images were acquired using LSM 900 equipped with Airyscan2 (Carl Zeiss).

[0148] The results are shown in Figure 6 . Among them, it can be known from a in Figure 6 that there were a large number of mtDNA released from the damaged mitochondria in the CIRI lesions of the I / R group, and MSNO could significantly reduce the release of mtDNA, and its therapeutic effect was significantly better than MS; it can be known from b in Figure 7 that most of the microglia in the CIRI lesions of the I / R group were activated to M1 type, and the iNOS / CD206 ratio was 4.02. Surprisingly, the M1 phenotype of microglia in the MSNO group was reduced, and the M2 phenotype was increased, suggesting that MSNO could promote the transformation of microglia from M1 phenotype to M2 phenotype; it can be known from c-d in Figure 7 that the positive staining cells of cGAS and STING in the CIRI lesions of the I / R group were significantly increased, and MSNO significantly inhibited the activation of cGAS-STING, indicating that MSNO regulated the phenotype transformation of microglia by inhibiting the activation of cGAS-STING pathway; it can be known from Figure 7From Fig. 6, it was found that the expression levels of cGAS, STING and downstream molecules P-IRF3 / IRF3, P-P65 / P65 in the brain tissue of I / R model group were more than 2 times of those in the Sham group, confirming that the cGAS-STING signaling pathway was significantly activated in CIRI. After MSNO treatment, it could significantly reduce the high expression of cGAS and STING and the phosphorylation levels of IRF3 and P65 induced by I / R, and could significantly reduce the release of pro-inflammatory factors (IL-1β, IL-6, iNOS and TNF-α) and increase the secretion of anti-inflammatory factors (IL-4 and IL-10) induced by I / R. In summary, from Fig. 6, it was found that the expression levels of cGAS, STING and downstream molecules P-IRF3 / IRF3, P-P65 / P65 in the brain tissue of I / R model group were more than 2 times of those in the Sham group, confirming that the cGAS-STING signaling pathway was significantly activated in CIRI. After MSNO treatment, it could significantly reduce the high expression of cGAS and STING and the phosphorylation levels of IRF3 and P65 induced by I / R, and could significantly reduce the release of pro-inflammatory factors (IL-1β, IL-6, iNOS and TNF-α) and increase the secretion of anti-inflammatory factors (IL-4 and IL-10) induced by I / R. In summary, Figure 7 From Fig. 6, it was found that the expression levels of cGAS, STING and downstream molecules P-IRF3 / IRF3, P-P65 / P65 in the brain tissue of I / R model group were more than 2 times of those in the Sham group, confirming that the cGAS-STING signaling pathway was significantly activated in CIRI. After MSNO treatment, it could significantly reduce the high expression of cGAS and STING and the phosphorylation levels of IRF3 and P65 induced by I / R, and could significantly reduce the release of pro-inflammatory factors (IL-1β, IL-6, iNOS and TNF-α) and increase the secretion of anti-inflammatory factors (IL-4 and IL-10) induced by I / R. In summary, from Fig. 6, it was found that the expression levels of cGAS, STING and downstream molecules P-IRF3 / IRF3, P-P65 / P65 in the brain tissue of I / R model group were more than 2 times of those in the Sham group, confirming that the cGAS-STING signaling pathway was significantly activated in CIRI. After MSNO treatment, it could significantly reduce the high expression of cGAS and STING and the phosphorylation levels of IRF3 and P65 induced by I / R, and could significantly reduce the release of pro-inflammatory factors (IL-1β, IL-6, iNOS and TNF-α) and increase the secretion of anti-inflammatory factors (IL-4 and IL-10) induced by I / R. In summary,

[0149] Example 21

[0150] Example 1, the protective effect of the nanodrug on endoplasmic reticulum stress (ERS) and apoptosis in the infarct area of CIRI rats was evaluated. The specific steps are as follows:

[0151] Animal level: The apoptosis of brain infarct tissues in different groups was analyzed by tdt-mediated dUTP end labeling (TUNEL) staining. The expression of apoptosis-related proteins in brain tissues induced by ischemia / reperfusion (I / R) was detected by WB method. The detailed method of tissue section fluorescence staining and WB detection of protein level is the same as that of Example 20. The detailed method of tissue TEM is the same as that of Example 16.

[0152] Quantitative real-time PCR analysis: Total RNA was extracted from frozen brain tissue / cells using TRIzol and reverse transcribed to obtain cDNA. Quantitative real-time PCR was performed using Applied Biosystems Step One Plus instrument and TB Green Premix Ex TaqTM(TliRNaseH Plus). The method of comparing Ct values was used to evaluate gene expression, and β-Actin was used as a reference gene.

[0153] Cell level: SH-SY5Y cells were seeded at 1×10 5Cells were seeded at a density of [insert density here] in 6-well plates. After cell adhesion, cells in the normoxic group were incubated normally. Cells in the H / R model group were incubated in culture medium containing 400 μM cobalt chloride for 16 h, then replaced with normal culture medium and incubated for another 8 h. Cells in the drug-treated group were incubated in culture medium containing 400 μM cobalt chloride and MS / MSNO for 16 h, then incubated in culture medium containing only MS / MSNO for another 8 h. Subsequently, cells from each group were collected, treated according to the Annexin V-FITC / PI reagent instructions, and then analyzed by flow cytometry.

[0154] The results are as follows Figure 7 As shown. Among them, by Figure 7 As observed in the CT scan, the endoplasmic reticulum of neurons in the I / R group showed significant swelling along with mitochondrial destruction, confirming severe endoplasmic reticulum (ERS) in CIRI neurons. In contrast, the MSNO group showed significantly improved endoplasmic reticulum edema, with a structure similar to the Sham group, indicating that MSNO can effectively improve neuronal ERS in CIRI. Figure 7 As shown in section d, neurons are extremely sensitive to the rate of protein synthesis. mtROS causes misfolding of neuronal proteins in CIRI. These misfolded proteins bind to Bip on the endoplasmic reticulum membrane, activating three transmembrane ERS molecules: inositol-requiring enzyme 1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK), and activating transcription factor 6 (ATF-6), thereby inducing ERS and further promoting apoptosis. Specifically, PERK phosphorylates eukaryotic initiator factor-2α (eIf-2α), increasing the expression of transcription factors such as activating transcription factor 4 (ATF-4). ATF-6 is transported to the Golgi apparatus, where it is cleaved by endopeptidases S1P and S2P, releasing the cytoplasmic ATF-6 fragment. ATF-4 and ATF-6 initiate Chop transcription, promoting neuronal apoptosis. IRE1 activates the transcription factor X-box binding protein (XBP1), catalyzing the JNK and Caspase-12-mediated apoptosis signaling pathway. Figure 6 As shown in the results, Bip expression was significantly increased in I / R brain tissue and H / R SH-SY5Y cells, indicating that ER stress is triggered both in vivo and in vitro. Figures 8-10 As shown in fh, PERK, ATF6, and IRE-1α were significantly activated, further stimulating the expression of downstream factors, including eIf-2α, ATF-4, Chop, XBP-1s, JNK, and Caspase 12. Since MSNO can effectively eliminate mtROS, it can inhibit ERS and almost completely reverse the activation of these pathways. ​ As can be seen from the results, the apoptosis rate of brain tissue cells in the I / R group was as high as 74.75%, while the apoptosis rate in the MSNO treatment group (9.05%) was significantly reduced. The MS group showed some effect (30.94%), but the effect was not as good as that in the MSNO group.​ As shown in Fig. 6, the expression levels of pro-apoptotic proteins Bax, cleaved-caspase3 / caspase3 (C-Cas / Cas), cytoplasmic-cytochrome C (C-Cyt C) in the I / R group were significantly increased, which were 3.75, 3.57, 2.3 times of those in the Sham group, respectively, and the expression of anti-apoptotic factor Bcl-2 was significantly decreased, which was 0.58 times of that in the Sham group. Both MS and MSNO could significantly reverse the expression of the above proteins, and the treatment effect of MSNO was significantly better than that of MS. Further, flow cytometry was used to detect the improvement of MSNO on the apoptosis of SH-SY5Y cells induced by H / R. As shown in Fig. 7, the apoptosis rate of SH-SY5Y cells induced by H / R was as high as 39.81%, and the apoptosis rate of cells in the MS treatment group was reduced to 24.25%, and the apoptosis rate of cells in the MSNO treatment group was reduced to 12.30%, indicating that MSNO could also effectively inhibit the apoptosis of neurons induced by H / R in vitro. In summary, as shown in Fig. 8, MSNO could effectively alleviate ERS and significantly reduce the apoptosis of neurons, and the effect was much better than that of MS which could only remove ROS. ​ ​ As shown in Fig. 8, MSNO could effectively alleviate ERS and significantly reduce the apoptosis of neurons, and the effect was much better than that of MS which could only remove ROS.

[0155] Example 22

[0156] The nano-drug 1 (MSNO) synthesized in Example 1 was taken as an example, and the biocompatibility of the nano-drug was evaluated at the cell level and the animal level, respectively. The specific steps are as follows:

[0157] Animal level: 24 hours after healthy SD rats were given 5 mg / kg of MSNO nano-drug (dissolved in normal saline) by tail vein injection, the rats were euthanized and the main organs (brain, heart, liver, spleen, lung, kidney) were collected. The paraffin sections of each tissue organ were prepared to observe the tissue morphology by hematoxylin-eosin staining. Specifically, after the paraffin sections were deparaffinized, hematoxylin staining solution was added dropwise to the tissues to ensure complete coverage of the tissues, and the tissues were stained for 10-15 minutes. The excess dye was washed away by flowing water. 1% hydrochloric acid alcohol solution was differentiated, and the tissues were soaked in water for 2 minutes to return to blue. Eosin staining solution was added dropwise on the tissues for about 10 seconds, and after the eosin staining was completed, the tissues were immediately immersed in anhydrous ethanol for dehydration twice (2 minutes each time). The tissues were immersed in xylene twice (2 minutes each time), and then naturally air-dried in a fume hood for 10-30 minutes. The tissues were mounted with neutral resin, and then observed under a microscope and the images were collected. Blood samples were taken to evaluate the liver function related indicators (alanine transaminase (ALT) and gamma-glutamyl transferase (γ-GT)), kidney function indicators (creatinine (CR) and urea nitrogen (BUN))

[0158] Cell level: SH-SY5Y cells and BV2 cells were seeded in 6-well plates at a concentration of 1×10 4 ​The cells were seeded into 96-well plates at a density of 1 well, and incubated for 24 hours. MSNO was dispersed in the culture medium to prepare cell culture solutions at different concentrations (0, 0.25, 0.5, 1, 5, 2, 4, 8, 16, 32 and 64 μg / mL, respectively). The cell culture solutions were added to SH-SY5Y cells and BV2 cells, and incubated for 24 / 48 hours. Then, 10 μL of CCK-8 reagent was added to each well, and incubated for half an hour. Cell viability was detected by measuring absorbance at 450 nm.

[0159] The results are shown in Table 1. ​ The results are shown in Table 1.

[0160] Obviously, the above examples are merely illustrative and not intended to limit the embodiments. Based on the above description, other different forms of changes or variations can be made by those of ordinary skill in the art. All embodiments are not required to be exhaustive. The obvious changes or variations derived therefrom are still within the scope of the present application.

Claims

1. A nanomedicine with eNOS-like enzyme activity, characterized in that, It is a NO-intercalated molybdenum sulfide nanomaterial (MSNO) obtained by introducing S-nitroso (-SNO) groups on the surface of small molybdenum sulfide (MS) particles, wherein the S-nitroso groups are doubly connected to the surface of molybdenum sulfide through S-NO bonds and Mo-N coordination bonds; The particle size of the molybdenum sulfide is between 20 and 200 nm. The introduction of S-nitroso (-SNO) groups onto the surface of small-particle molybdenum sulfide (MS) is achieved by reacting small-particle molybdenum sulfide (MS) with sodium nitrite.

2. A method for preparing a nanomedicine with eNOS-like enzyme activity as described in claim 1, characterized in that, Includes the following steps: Ammonium molybdate tetrahydrate ((NH4)6Mo7O 24 MS was prepared by hydrothermal method using 4H2O and thiourea (CN2H4S); small particle MS was screened by differential centrifugation. Small MS particles were washed with hydrochloric acid and reacted with sodium nitrite to introduce S-nitroso (-SNO) groups onto the MS surface, resulting in NO-intercalated MSNO.

3. The method for preparing nanomedicines with eNOS-like enzyme activity according to claim 2, characterized in that, The reaction system prepared by MS is an alkaline buffer solution with pH=8-10, the reaction temperature is 180℃, and the reaction time is 7-24 hours; the reaction system with the introduction of S-nitroso (-SNO) group is 0℃, and the reaction time is 12-24 hours.

4. The method for preparing nanomedicines with eNOS-like enzyme activity according to claim 2, characterized in that, The mass ratio of ammonium molybdate tetrahydrate and thiourea is between 5:1 and 1:

5.

5. The method for preparing nanomedicines with eNOS-like enzyme activity according to claim 2, characterized in that, Differential centrifugation was used to screen small MS particles at speeds between 1000 rpm and 5000 rpm, with centrifugation times between 1 and 10 min. The concentration of hydrochloric acid used for washing the small MS particles ranged from 6 mol / L to 12 mol / L, and the washing time was between 12 and 24 h.

6. The method for preparing nanomedicines with eNOS-like enzyme activity according to claim 2, characterized in that, The mass ratio of MS to sodium nitrite is between 10:1 and 1:

10.

7. The use of the nanomedicine with eNOS-like enzyme activity as described in claim 1 in the preparation of a drug for treating cerebral ischemia-reperfusion injury.

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